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Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin-based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo-sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo-resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26-27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo- resistance to cisplatin-based treatment in TGCTs and other cancers. © 2008 Wiley-Liss, Inc.

Purpose: To assess the safety and toxicity profile of escalating doses of intravenous irinotecan, in combination with a fixed dose of oral ciclosporin (Cs) and to determine the pharmacokinetic profile of irinotecan and its metabolites. Patients and Methods: Patients with fluorouracil-refractory metastatic colorectal cancer received escalating doses of intravenous irinotecan from 40 to 125 mg/m2 every 2 weeks in combination with a fixed dose of oral Cs (5 mg/kg bid for 3 days). Pharmacokinetic analysis of plasma irinotecan and its metabolites SN38 and SN38G was performed during paired cycles with and without Cs. Results: Thirty-seven patients were treated. Dose-limiting toxicity of grade 4 neutropenia was seen at an irinotecan dose of 125 mg/m2. There was no grade 4 diarrhea, and only one patient experienced grade 3 diarrhea. Toxicities caused by Cs were generally mild. Pharmacokinetic studies demonstrated that irinotecan clearance was reduced from 13.4 to 5.8 L/h/m2 and area under the curve (AUC)0-tn was increased 2.2-fold by the coadministration of Cs. Similar significant increases in AUC0-24h were seen for both SN38 and SN38G (2.2-fold and 2.3-fold, respectively) in the presence of Cs. Antitumor activity was seen at every irinotecan dose level. Conclusion: The maximum tolerated irinotecan dose and recommended dose for phase II studies is 100 mg/m2 every 2 weeks. Dose-limiting diarrhea was not seen during this study, supporting the hypothesis that pharmacokinetic modulation of irinotecan by Cs may improve its therapeutic index. Further studies using this combination are warranted.

Lapatinib is a specific HER1 and 2 targeted tyrosine kinase inhibitor now widely used in combination with chemotherapy in the clinical setting. In this work, we investigated the interactions between lapatinib and specific chemotherapy agents (cisplatin, SN-38, topotecan) in a panel of cell lines [breast (n = 2), lung (n = 2), testis (n = 4)]. A high-sensitivity cell proliferation/cytotoxicity ATP assay and flow cytometry were used to determine cell viability, apoptosis, and the effect of the drugs on cell-cycle distribution. CalcuSyn analysis was employed to formally identify synergistic interactions between drugs. Intracellular concentrations of SN-38 were measured using a novel high-performance liquid chromatography (HPLC) technique. Flow cytometry and HPLC techniques were used to identify the effect of lapatinib on drug influx and efflux pumps, using specific substrates and inhibitors of these pumps. Results showed significant synergy between SN-38, and lapatinib in the majority of cell lines (combination index < 0.75), associated with increased apoptosis. This synergy was not universal but, when observed (Susa S/R, H1975, H358, and MDA-MB-231 cell lines), was related to SN-38 intracellular accumulation (2.2- to 4.8-fold increase, P < 0.05 for each), attributable to the inhibition of the breast cancer–related protein (BCRP) efflux pump by lapatinib. Flow cytometry analysis showed that lapatinib (10 μmol/L) inhibited the efflux of mitoxantrone, a specific substrate of the BCRP pump, in a manner similar to fumitremorgin C, a known BCRP inhibitor, confirming lapatinib as a BCRP inhibitor. This work shows that lapatinib has a direct inhibitory effect on BCRP accounting for the synergistic findings. The synergy is cell line dependent and related to the activity of specific efflux pumps. Mol Cancer Ther; 9(12); 3322–9. ©2010 AACR.

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Germ cell tumours (GCT) can become resistant to cisplatin, which is associated with a relatively poor prognosis. Oxaliplatin and satraplatin have been developed to overcome cisplatin resistance in other cancers, but their effect in cisplatin resistant (cisR) GCTs is unclear. In this work we address this issue by comparing their efficacy in three paired sensitive and cisR GCT cell lines.Three established cisplatin sensitive (cisS) and resistant cell line pairs were used (GCT27, GCT27r: SUSA, SUSAr: 833k, 833kr). Viability was assessed using a luciferase based ATP assay and EC(50) and EC(80) concentrations were calculated. Western blot analysis and flow cytometry was used for further assessment.Sensitivity to the three platinum compounds was broadly similar in the three cisS lines GCT cell lines (EC(50) = 0.27-0.51 microM for cisplatin, 0.52-0.79 microM for oxaliplatin, 0.31-1.26 microM for satraplatin). EC(50) values for cisplatin in the three cisR sub lines were 1.8- to 3.8-fold higher than in the sensitive parental lines. Cross resistance to satraplatin and oxaliplatin occurred in all three cisR cell lines (resistance factor 1.9-4.4), with the exception of oxaliplatin in the 833Kr (resistance factor 0.9). Differences in the effect of specific drugs on cell cycle distribution, p53, p21 and MDM2 were observed.These data suggest that satraplatin and oxaliplatin could theoretically be used in chemo-naive GCTs and support the further clinical evaluation of these agents in this setting. The mechanism of cross resistance to these drugs appears multifactorial.